The long-term goal of this research is a detailed understanding of the molecular mechanisms of gene regulation in higher organisms. Specifically, I propose to apply recently developed methods for the selective and accurate synthesis of RNA in cell-free systems to the problem of control of transcription. First, more detailed studies will be done on accurate RNA synthesis in vitro, in order to understand separately both the initiation and elongation reactions as a basis for later experiments. Second, a series of in vitro mutagenized promotors will be transcribed in the cell-free system, to determine what DNA sequences are required for a functional promotor. Third, in order to assess the effect of packaging the template DNA into nucleosomes, promotor-bearing DNA fragments will be assembled in vitro into model chromatin structures and then transcribed in the cell-free system. Fourth, an attempt will be made to demonstrate physiologically meaningful regulation of cell-free transcription, using hemoglobin genes as a test system. Nuclear proteins will be prepared by several different methods from cultured cells which synthesize globin mRNA or from non-globin producing control cells; such proteins will be reconstituted with purified globin DNA or globin DNA containing minichromosomes. These reconstitutes will be assayed in the cell-free transcription system in order to identify these components from globin-producing cells which modulate globin transcription in the expected way, but which do not affect the transcription of control (non-globin) genes.